Two distinct signals are required for the activation of T cells. The first is an antigen-specific interaction between the T cell receptor (TCR) and nominal antigen presented in the context of the MHC on the surface of an antigen-presenting cells (APCs). The second signal is provided through a number of potential co-stimulatory molecules. The activation of T cell is tightly regulated by multiple mechanisms, including cell surface proteins which expand or downregulate T cell responses (Bretscher et al., (1970) Science 69: 1042; Bernard et al., (2002) Transplantation 73: S31-S35). CD28, a constitutively expressed Ig-family protein, is one of the best-characterized co-stimulatory signals for T cell response. CD28 binding to ligands CD80 (B7-1) and CD86 (B7-2) on APCs leads to T cell proliferation by inducing production of interleukin-2 (IL-2) and anti-apoptotic factors. CTLA4 is the first molecule identified as a co-inhibitory molecule and play an important role in regulating both humoral and cellular immune response (Brunet et al., (1987) Nature 328:267-270). CTLA4 belongs to CD28 superfamily with 31% overall amino acid identity to CD28. CTAL4 is composed of disulfide-linked homodimers of extracellular IgV domains. (Stamper et al., (2001) Nature 410: 608-611). Unlike other inhibitory receptors, CTLA4 lacks a classic immunoreceptor tyrosine-based inhibitory motif (ITIM). Despite this, two phosphatases, SHP-2 and the serine-threonine phosphatase protein phosphatase 2A (PP2A), have been reported to associate with the YVKM motif of CTLA4 (Rudd et al., (2009) Immunol Rev. 229: 12-26). CD28 and CTLA4 share CD80 and CD86 as their natural ligands. However, the affinity of the CTLA4:B7 interaction is over 10 times higher than the affinity of the CD28:B7 interaction (Peach et al., (1994) J Exp Med 180:2049-2058). This allows CTLA4 to sequester B7 ligands from CD28 and antagonize CD28-dependent costimulation, which account for part of the inhibitory effect of CTLA4 on T cell activation. CTLA4 has also been proposed to deliver distinct distal signals independent of the TCR signal to attenuate T cell responses (Calvo et al., (1997) J Exp Med 186: 1645-1653). By interacting with the B7 molecules on APCs, CTLA4 induces the expression of IDO, which catalyzes the conversion of tryptophan to kynurenine, resulting in a local tryptophan depletion and subsequent inhibition of T cell proliferation and activation (Mellor et al., (2004) Int Immunol 16: 1391-1401). Lipid raft-associated CTLA4 interacted intimately with the TCR complex and altered lipid raft integrity and TCR-mediated signals (Chikuma et al., (2003) J Exp Med 197: 129-135). In addition, CTLA4 can function independent of B7 ligation as a consequence of recruitment to the synapse on activated T cells (Chikuma et al., (2005) J Immunol 175: 177-181).
The importance of CTLA4 as a negative regulator is dramatically revealed through the phenotype of CTLA4 knockout mice (Tivol et al., (1995) Immunity 3:541-547). CTLA4 deficient mice develop a massive and rapidly lethal T-lymphoproliferative disease with splenomegaly, lymphadenopathy and multiorgan T-lymphocytic infiltration, resulting from excessive proliferation of T cells following recognition of antigen and unopposed or uncompeted co-stimulatory interactions between CD80/CD86 and CD28. In addition, polymorphisms in the CTLA4 gene are linked with several autoimmune diseases (Gough et al., (2005) Immunol Rev 204:102-15), including type 1 diabetes, thyroiditis, systemic lupus erythematosus, and rheumatoid arthritis.
While CD28 is expressed on most resting and activated T cells, CTLA4 is restricted to activated T cells, except in the case of regulatory T cells (Treg) where it is expressed constitutively. CTLA4 functions on both Treg and CD8 effector cells (Teff). CTLA4 targets the transcription factor Eomes in the regulation of CD8+ effector function, and results in reduced IFNγ and Granzyme B expression and potential cytolytic T-cell function. Loss of CTLA4 expression on Treg cells impairs their suppressive function and elicit pathological autoimmunity. The inhibitory effect on Teff and the stimulatory effect on Treg of CTLA4 lead to attenuated immune responses, and thus mediates tolerance and/or anergy (Carreno et al., (2000) J Immunol 165: 1352-1356; Chai et al., (2000) J Immunol 165: 3037-3042).
CTLA4 has been found to have a correlation with cancer growth and development due to its negative role in immune response. CTLA4 is expressed in tumors at higher levels on Treg cells as compared with intra-tumoral Teff cell, and it has been shown that anti-CTLA4 needs to bind to Treg cells and to Teff cells to induce full tumor protection (Peggs et al., (2009) J Exp Med 206: 1717). Furthermore, anti-CTLA4-mediated tumor destruction was regularly associated with an increased ratio of intra-tumoral CD4+ Teff/Treg cells and an increased ratio of intra-tumoral CD8+ Teff/Treg cells (Quezada et al., (2006) J Clin Invest 116: 1935; Curran et al., (2010) Proc Natl Acad Sci USA 107: 4275).
In early studies with animal models, antibody blockade of CTLA4 was shown to exacerbate autoimmunity (Perrin et al., (1996) J Immunol 157: 1333-6; Hurwitz et al., (1999) J Neuroimmunol 73: 57-62). By extension to tumor immunity, blockade of the CTLA4 inhibitory signal was accordingly shown to enhance tumor-specific T-cell immunity and cause regression of established tumors. In a murine model of aggressive colon cancer, for example, Leach et al. demonstrated the therapeutic efficacy of CTLA4 blockade. Administration of CTLA4 directed antibody significantly rejected tumor growth of both CD80 positive and CD80 negative colon carcinoma. Furthermore, this rejection resulted in immunity to a secondary exposure to tumor cells. Additionally, the authors showed that treatment with anti-CTLA4 also reduced the growth of the murine fibrosarcoma Sa1N (Leach et al., (1996) Science 271: 1734-1736). Recent studies suggested that direct enhancement of Teff cell function and concomitant inhibition of Treg cell activity through blockade of CTLA4 on both cell types is essential for mediating the full therapeutic effects of anti-CTLA4 antibodies during cancer immunotherapy (Peggs et al., (2009) J Exp Med 206:1717-25).
The versatility of CTLA4 blockade, in combination with multiple therapeutic interventions, has been reported in a variety of mouse tumor models, such as 4T1 (breast cancer), EL4 (lymphoma), CT26 (colon cancer) (Jure-Kunkel et al., (2008) J Clin Oncol 26 Suppl 15: 3048). Synergistic effects on anti-tumor activity have been demonstrated in combination with vaccines (Saha et al., (2010) Scand J Immunol 71: 70-82), chemotherapy (Mokyr et al., (1998) Cancer Res 58: 5301-5304), radiation (Dewan et al., (2009) Clin Cancer Res 15: 5379-5388), cytosine-phosphateguanine oligodeoxynucleotides (CpG-ODN) adjuvants (Davila et al., (2003) Cancer Res 63: 3281-3288), antibodies (Takeda et al., (2010) J Immunol 184: 5493-5501; Redmond et al., (2013) Cancer Immunol Res 2: 142-53) and cryoablation (Waitz et al., (2012) Cancer Res 72: 430-439.). For an example, using 3 different tumor lines: SAIN fibrosarcoma, M109 lung carcinoma and EMT-6 mammary carcinoma, Jure-Kunkel et al demonstrated that the combination of the anti-CTLA4 antibody and ixabepilone showed a synergistic antitumor effect in these tumor models achieving long-lasting complete responses in 70-100% of the animals, which yielded much superior efficacy compared to each treatment alone. When animals with complete tumor regressions were rechallenged with a lethal dose of tumor cells, animals treated with ixabepilone plus CTLA4 antibody rejected a subsequent tumor, indicating the development of a protective memory immune response (Jure-Kunkel et al., (2008) J Clin Oncol 26 Suppl 15: 3048).
Ipilimumab, a human anti-CTLA4 antibody capable to block CTLA4/B7 interactions (Keler et al., (2003) J Immunol 171: 6251-9) has been tested in a variety of clinical trials for multiple malignancies (Hoos et al., (2010) Semin Oncol 37: 533-46; Ascierto et al., (2011) J Transl Med 9: 196). Tumor regressions and disease stabilization were frequently observed, and accompanied by adverse events with inflammatory infiltrates capable of affecting a variety of organ systems. In 2011, ipilimumab, was approved for the treatment of unresectable or metastatic melanoma in the United States and European Union based on an improvement in overall survival in a phase III trial of previously treated patients with advanced melanoma (Hodi et al., (2010) N Engl J Med 363: 711-23).
Ipilimumab treatment, however, has been associated with severe and potentially fatal immunological adverse effects due to T cell activation and proliferation in 10-20% of people being treated. The cost of ipilimumab treatment is staggering high. Therefore, there is continuing need to develop novel antibodies against CTLA4.